Motor proteins operate in a complex environment in the cell where interactions with motors of the same type, oppositely-directed motors, and the cytoskeletal landscape allow long-range, bidirectional transport. Interactions among motor proteins and with the cytoskeleton modulate the behavior of motor proteins in vivo and make possible the targeted trafficking of intracellular cargoes. Motor proteins of different types often associate simultaneously with vesicular cargoes to allow bidirectional transport along microtubules and switching between microtubules and actin filaments. The objective of this study is to elucidate the dynamics of bidirectional transport through analysis of the interactions among motor proteins and with the cytoskeleton in increasingly complex in vitro approximations of the physiological environment. Specifically, we propose the following aims: 1. To investigate the mechanism of interaction between the oppositely-directed motors kinesin and dynein when transporting artificial cargoes. Is bidirectional transport by opposing motors coordinated through regulatory proteins, or the result of a tug-of-war between oppositely-directed motors? How multiple kinesin or dynein do motors function collectively in teams? What role do Microtubule Associated Proteins (MAPs) have in regulating bidirectional transport? 2. To examine bidirectional transport of purified endogenous vesicles and endocytosed cargoes. How does the endogenous complement of motors interact? What influence do the effectors that copurify with the vesicles have on motor function and coordination? 3. To develop a mechanistic, mathematical model of bidirectional transport by kinesin and dynein. A mathematical model will provide a feedback mechanism for the experiments, functioning to test our understanding of bidirectional transport and direct further experiments. What mechanisms of interactions among motors produce results consistent with experimental observations? What future experiments are best suited to further the understanding of bidirectional transport? By systematically increasing the complexity of in vitro assays, we can separate the influence of complicating factors (i.e. motor-motor interactions, cytoskeletal networks, and MAPs) and understand each independently. We can then integrate these individual aspects into more complex assays, sequentially incorporating aspects of the cellular environment. The integrated in vitro experiments and mathematical models will provide an understanding of the dynamics and regulation of intracellular transport, which is critical as defects in intracellular transport are strongly implicated in both developmental and degenerative diseases in humans. PUBLIC HEALTH RELEVANCE: The objective of this study is to elucidate the regulation and dynamics of motor proteins in intracellular transport through analysis of motors in increasingly complex in vitro approximations of the physiological environment. An understanding of intracellular transport is critical as defects are strongly implicated in both developmental and degenerative diseases in humans. For example, a missense mutation in dynactin leads to autosomal dominant motor neuron degeneration and mutations in kinesins have been linked to Charcot-Marie-Tooth Disease Type 2A and Hereditary Spastic Paraplegia.